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The significance of Howell-Jolly bodies and giant metamyelocytes in marrow smears.

Howell-Jolly bodies in erythroid precursors can be found in the marrow in a variety of diseases. More than 1% in the later erythroblasts is rarely found except in vitamin B(12) or folic acid deficiency states.Giant metamyelocytes are probably always the result of vitamin B(12) or folic acid deficiency. A distinction is drawn between the deficiency of, and therapeutic need for, vitamin B(12) and folic acid.

Bone Marrow↗

Flow cytometric study of neutrophilic granulopoiesis in normal bone marrow using an expanded panel of antibodies: correlation with morphologic assessments.

Flow cytometry studies of surface markers of neutrophils have been performed mostly on peripheral blood, and for a limited spectrum of diseases. Studying maturation defects on developing neutrophils in the bone marrow (BM) may be helpful in BM diseases, such as myelodysplastic syndromes and Shwachman-Diamond syndrome. We applied an expanded panel of antibodies to examine normal maturation patterns in 26 control samples of BM together with microscopic correlation. Promyelocytes correlated well with the CD24(-) and CD11b(-) populations, and metamyelocytes correlated well with the CD16(+) population (intermediate positivity). An excellent correlation was also identified between the sum of bands and segmented neutrophils and each of the following: CD16(++) (strong positivity), CD35(+), CD87(+), and CD64(-). Although visually identified segmented neutrophils paralleled CD10 positivity, there was an appreciable difference between both methods. We conclude that neutrophilic granulocyte maturation in the BM is accompanied by a change in surface antigens that reflects certain stages of development. A successful strategy for detecting maturation defects is to include several antibodies that are known to be expressed or absent at the same stage of maturation, such as CD16, CD35, CD64, and CD87.

Adolescent↗

Mitochondrial disease activates transcripts of the unfolded protein response and cell cycle and inhibits vesicular secretion and oligodendrocyte-specific transcripts.

Mutations in gene products expressed in the mitochondrion cause a nuclear transcriptional response that leads to neurological disease. To examine the extent to which the transcriptional profile was shared among 5 mitochondrial diseases (LHON, FRDA, MELAS, KSS, and NARP), we microarrayed mutant and control groups in N-tera2, SH-SY5Y, lymphoblasts, fibroblasts, myoblasts, muscle, and osteosarcoma cybrids. Many more transcripts were observed to be significantly altered and shared among these 5 mitochondrial diseases and cell types than expected on the basis of random chance, and these genes are significantly clustered with respect to biochemical pathways. Mitochondrial disease activated multiple transcripts of the unfolded protein response (UPR), and of the cell cycle pathway, and low doses of the mitochondrial inhibitor rotenone induced UPR transcripts in the absence of cell death. By contrast, functional clusters inhibited by mitochondrial disease included: vesicular secretion, protein synthesis, and oligodendrogenesis. As it is known that UPR activation specifically inhibits vesicular secretion and protein synthesis, these data support the view that mitochondrial disease and dysfunction triggers the UPR, which in turn causes secretory defects which inhibit cellular migratory, synaptic, and oligodendrocytic functions, providing a testable hypothesis for how mitochondrial dysfunction causes disease. Since ischemic hypoxia, chemical hypoxia, and mitochondrial genetic disease (which could be considered 'genetic hypoxia') produce an overlapping induction of UPR and cell cycle genes which appears to have negative consequences, the modulation of these responses might be of benefit to patients with mitochondrial disease.

Cell Cycle Proteins↗

Effect of 1,25(OH)2 Vitamin D3 analogs on differentiation induction and cytokine modulation in blasts from acute myeloid leukemia patients.

In acute myeloid leukemia (AML), cell proliferation and differentiation are uncoupled, causing a maturation block. Induction of terminal differentiation is a potential therapeutic strategy. 1alpha, 25(OH)2 Vitamin D3 regulates differentiation and is immunomodulatory at concentrations causing severe hypercalcemia, thus limiting its use. We investigated 1alpha, 25(OH)2 Vitamin D3 and 5 of its more potent analogs with reduced calcium resorbing activity for differentiation of blast cells from AML (FAB M1) patients, compared to TPA. Blast phenotype, p-glycoprotein expression, cytokine production, and lineage specificity were examined. The Vitamin D3 analogs had no effect on cell viability and proliferation. They induced incomplete differentiation, with increase in AP, NSE and NBT positivity of cells, but no cell sticking and spreading as observed with TPA. The analogs were more effective than the parent compound. They also inhibited the production of IL-6 and IL-8. Vitamin D3 and its analogs can induce differentiation of primary cells from AML patients in vitro, but may need to be combined with other agents for terminal differentiation of blasts and effective therapy in vivo.

Acute Disease↗

Overexpression of interleukin 21 induces expansion of hematopoietic progenitor cells.

The interleukin 21 (IL-21) receptor is expressed on T-cells, B-cells, and natural killer cells, and IL-21 is critical for regulating immunoglobulin production in vivo in cooperation with IL-4. So far, however, little is known about a role for IL-21 outside the immune system. We investigated the effect of IL-21 on hematopoiesis in vivo by using the hydrodynamics gene-delivery method. Overexpression of IL-21 increases Sca-1+ cells in the periphery and spleen. It also increases the numbers of c-Kit+, Sca-1+, and lineage-/low (KSL) cells and colony-forming units-granulocyte-macrophage (CFU-GM) in the spleen, indicating the expansion of hematopoietic progenitor cells. We found that even in RAG2-/- mice, which lack mature T-cells and B-cells, IL-21 induced an increase in KSL cells and CFU-GM in the spleen. These results demonstrate that IL-21 can induce the expansion of hematopoietic progenitor cells in vivo, even in the absence of mature T-cells and B-cells.

Animals↗

Two methods for the quantitative analysis of surface antigen expression in acute myeloid leukemia (AML).

The expression of lineage molecules (CD13 and CD33), c-Kit receptor (CD117), CD34, HLA-DR and adhesion molecule CD49d was assessed in acute myeloid leukemia (AML) blast cells from 32 cases, using direct and indirect quantitative cytometric analysis. High correlation (r=0.8) was found between antigen expression intensity values calculated by direct analysis method (ABC) and by indirect analysis method (RFI). Moreover, the differences in expression intensity of CD13, CD117 and CD34 antigens were found between leukemic and normal myeloblasts. This may be helpful in identification of leukemic cells in the diagnostics of minimal residual disease after treatment in AML patients.

Adult↗

The effect of stem cell factor on irradiated human bone marrow.

This study evaluates the effect of recombinant human stem cell factor (SCF) on the in vitro response of human bone marrow progenitor cells to irradiation. Light density nonadherent mononuclear cells were isolated from human bone marrow and resuspended in either semisolid culture or liquid culture with or without 100 ng/ml SCF. After 24 h in culture, cells were irradiated and assessed for survival of erythroid burst-forming unit, granulocyte colony-forming unit(s), or granulocyte-macrophage colony-forming unit precursors in the presence of erythropoietin, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor, respectively. Incubation with SCF prior to irradiation (0-300 cGy) resulted in an increase in both absolute colony number and surviving fraction for erythroid burst-forming units, granulocyte colony-forming units, and granulocyte-macrophage colony-forming units as compared to cultures that did not contain SCF. The mean surviving fraction enhancement ratio after 100 cGy ranged from 1.2 to 3.7. An increased fraction of CD34+ progenitors in S-phase after exposure to SCF may explain in part the apparent radioprotective effect of SCF on human bone marrow progenitor cells.

Bone Marrow↗

Interference of BCR-ABL1 kinase activity with antigen receptor signaling in B cell precursor leukemia cells.

The chromosomal translocation t,(9;22) resulting in the fusion of the BCR and ABL1 genes, represents a recurrent aberration in B cell precursor leukemia cells. Their normal counterparts, B cell precursor cells, are positively selected for survival signals through the antigen receptor, whose expression requires a functional immunoglobulin heavy chain (IGH) gene rearrangement. Unexpectedly, B cell precursor leukemia cells harboring a BCR-ABL1 gene rearrangement do not depend on antigen receptor mediated survival signals. Genes involved in the signaling cascade of the antigen receptor are silenced and in most cases, the dominant tumor clone does not carry a functional IGH gene rearrangement. However, upon inhibition of the BCR-ABL1 kinase activity by STI571, only leukemia cells expressing an antigen receptor are able to survive. Since resistance to STI571 is frequent in the therapy of BCR-ABL1(+) B cell precursor leukemia, antigen receptor signaling may represent a mechanism through which these cells can temporarily evade STI571-induced apoptosis. This may open a time frame, during which leukemia cells acquire secondary transforming events that confer definitive resistance to STI571.

Animals↗

Identification of a Myb-responsive enhancer of the chicken C/EBPbeta gene.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) that disrupts myelomonocytic differentiation and transforms myelomonocytic cells. It is thought that the biological effects of v-Myb are caused by deregulation of specific target genes. The CCAAT box/enhancer binding protein beta (C/EBPbeta), a member of the basic region-leucine zipper (bzip) class of transcription factors, which itself plays an important role during myelomonocytic differentiation, has previously been shown to be regulated by Myb. Here we have addressed the mechanism by which v-Myb affects C/EBPbeta expression. We have employed the mapping of DNase I hypersensitive sites (DHSs) in chromatin as a tool to detect in vivo target sites of v-Myb. Our data identify a DHS downstream of the C/EBPbeta gene that appears to be specific for v-myb-transformed myeloblasts. We have confirmed by chromatin immunoprecipitation that v-Myb is bound to this region in vivo. Furthermore, we have found that ectopic expression of v-Myb in a myelomonocytic cell line is able to induce a DHS downstream of the C/EBPbeta gene, showing for the first time that v-Myb can affect chromatin structure. Reporter gene experiments demonstrate that the downstream DHS acts as a Myb-dependent enhancing element in transiently as well as in stably transfected myelomonocytic cells. Previous work has shown that v-Myb acts on the C/EBPbeta promoter; it now appears that Myb stimulates C/EBPbeta expression by acting on the promoter as well as on an enhancer of the C/EBPbeta gene. Interestingly, the mechanisms by which Myb acts on both elements differ; while Myb activation of the promoter requires the cooperation with C/EBPbeta, activation of the enhancer by Myb is independent of C/EBPbeta. Apart from the identification of a novel Myb-dependent enhancer, our work demonstrates the potential of chromatin structure analysis for the identification of Myb target sites.

3' Flanking Region↗

The antiapoptotic gene A1/BFL1 is a WT1 target gene that mediates granulocytic differentiation and resistance to chemotherapy.

Previous work has demonstrated that WT1 (-Ex5/-KTS) potentiates granulocyte colony-stimulating factor (G-CSF)-mediated granulocytic differentiation. This WT1 isoform suppresses cyclin E, which may contribute to the prodifferentiation effect by slowing proliferation, but WT1 target genes that affect survival might also be involved. We screened a cDNA array and identified the bCL2 family member A1/BFL1 as a new WT1 target gene in 32D cl3 murine myeloblast cells. Induction of WT1 (-Ex5/-KTS) expression is accompanied by up-regulation of A1 on the cDNA array, and this up-regulation was confirmed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Moreover, both promoter-reporter assays and chromatin immunoprecipitation assays suggest that this isoform of WT1 activates the promoter directly. Constitutive expression of A1 in 32D cl3 cells induces spontaneous granulocytic differentiation, with both morphologic and cell-surface antigen changes, as well as resistance both to chemotherapy and to withdrawal of interleukin-3 (IL-3). Finally, we note an association between WT1 expression and A1 expression in primary acute myeloid leukemia samples. Taken together, these results demonstrate that A1 is a new WT1 target gene involved in both granulocytic differentiation and resistance to cell death, and suggests that these genes might play an important role in the biology of high-risk leukemias.

Animals↗

C/EBPalpha directs monocytic commitment of primary myeloid progenitors.

C/EBPalpha is required for generation of granulocyte-monocyte progenitors, but the subsequent role of C/EBPalpha in myeloid lineage commitment remains uncertain. We transduced murine marrow cells with C/EBPalpha-estradiol receptor (ER) or empty vector and subjected these to lineage depletion just prior to culture in estradiol with myeloid cytokines. This protocol limits biases due to lineage-specific effects on developmental kinetics, proliferation, and apoptosis. Also, lowering the dose of estradiol reduced activated C/EBPalpha-ER to near the physiologic range. C/EBPalpha-ER increased Mac1(+)/Gr1(-)/MPO(-)/low monocytes 1.9-fold while reducing Mac1(+)/Gr1(+)/MPO(hi) granulocytes 2.5-fold at 48 hours, even in 0.01 microM estradiol. This pattern was confirmed morphologically and by quantitative polymerase chain reaction (PCR) assay of lineage markers. To directly assess effects on immature progenitors, transduced cells were cultured for 1 day with and then in methylcellulose without estradiol. A 2-fold increase in monocytic compared with granulocytic colonies was observed in IL-3/IL-6/SCF or GM-CSF, but not G-CSF, even in 0.01 microM estradiol. C/EBPalpha-ER induced PU.1 mRNA, and PU.1-ER stimulated monocytic development, suggesting that transcriptional induction of PU.1 by C/EBPalpha contributes to monopoiesis. A C/EBPalpha variant incapable of zippering with c-Jun did not induce monopoiesis, and a variant unable to bind NF-kappaB p50 stimulated granulopoiesis, suggesting their cooperation with C/EBPalpha during monocytic commitment.

Animals↗

[Effects of Chinese herbs for supplementing Shen and strengthening bone on IL-6 mediated myelogenic osteoclasts formation of ovariectomized rats in early stage].

OBJECTIVE: To observe the effect of Chinese herbs for supplementing Shen and strengthening bone (HB) on myelogenic osteoclasts formation, and gene expression of interleukin-6 (IL-6), IL-6 receptor (IL-6R) and gp130 in bone marrow. METHODS: Seventy-two healthy female SD rats of 3 months, were randomly divided into three groups, 24 in the sham-operated group (A), 24 in the ovariectomized group (B) and 24 in the after ovariectomy HB treated group (C). Bone marrow cells of 6 rats from each group were respectively collected and cultured at four time points (2nd, 4th, 6th and 12th weeks after operation). After 6 days of culture, the bone marrow cells were differentiated by Wright-Giemsa stain and TRAP stain, and total RNA in them was extracted by TRIZOL. RESULTS: Beginning from the 2nd week, the osteoclasts formation in Group B was higher than that in Group A (P < 0.05), and IL-6, IL-6R gene expression significantly increased in Group B (P < 0.05 or P < 0.01). These changes reached the peak in the 4th to 6th week, with the level maintained to the 12th week. As for comparison of Group B and C, the above-mentioned changes were significantly weakened in the latter (P < 0.05 or P < 0.01). No significant change of gp130 gene expression revealed in the whole course in either group. CONCLUSION: HB could inhibit the myelogenic osteoclasts formation in ovariectomized rats, this effect may be correlated with, partially at least, its inhibitory effect on the over-expressed IL-6 and IL-6R gene expression in myelocytes after ovariectomy.

Animals↗

Short-term tests for transplacentally active carcinogens. Induction of sister-chromatid exchanges in foetal brain, lung and blood-forming cells by procarbazine and cyclophosphamide.

The induction of sister-chromatid exchanges (SCEs) by cyclophosphamide (CP) and procarbazine (PC) in mouse granulocyte-macrophage precursor cells (GM cells) and erythroblasts from foetal liver, and cells from foetal brain and foetal lung has been measured. Agents were administered in vivo, and cells explanted into BrdUrd-containing medium for 2 cell cycles in vitro (using specific growth-promoting substances where necessary) to determine SCE frequency. Tissue and cell-type differences in responses were observed, and it is concluded that the in vivo/in vitro transplacental SCE technique is a useful indicator of genotoxic effects of agents which are potential transplacental carcinogens.

Animals↗

High dexamethasone concentration prevents stimulatory effects of TNF-alpha and LPS on IL-6 secretion from the precursors of human muscle regeneration.

A frequent finding in patients surviving critical illness myopathy is chronic muscle dysfunction. Its pathogenesis is mostly unknown; one explanation could be that muscle regeneration, which normally follows myopathy, is insufficient in these patients because of a high glucocorticoid level in their blood. Glucocorticoids can prevent stimulatory effects of proinflammatory factors on the interleukin (IL)-6 secretion, diminishing in this way the autocrine and paracrine IL-6 actions known to stimulate proliferation at the earliest, myoblast stage of muscle formation. To test this hypothesis, we compared the effects of major proinflammatory agents [tumor necrosis factor (TNF)-alpha and endotoxin lipopolysaccharide (LPS)] on the IL-6 secretion from the muscle precursors and then studied the influence of dexamethasone (Dex) on these effects. Mononuclear myoblasts, which still proliferate, were compared with myotubes in which this capacity is already lost. For correct interpretation of results, cultures were examined for putative apoptosis and necrosis. We found that constitutive secretion of IL-6 did not differ significantly between myoblasts and myotubes; however, the TNF-alpha- and LPS-stimulated IL-6 release was more pronounced (P < 0.001) in myoblasts. Dex, applied at the 0.1-100 nM concentration range, prevented constitutive and TNF-alpha- and LPS-stimulated IL-6 release at both developmental stages but only at high concentration (P < 0.01). Although there are still missing links to it, our results support the concept that high concentrations of glucocorticoids, met in critically ill patients, prevent TNF-alpha- and LPS-stimulated IL-6 secretion. This results in reduced IL-6-mediated myoblast proliferation, leading to the reduced final mass of the regenerated muscle.

Cell Differentiation↗

Differential sensitivity of adherent CFU-blast, CFU-mix, BFU-E, and CFU-GM to mafosfamide: implications for adjusted dose purging in autologous bone marrow transplantation.

The availability of an in vitro assay able to detect hematopoietic progenitor cells closely related to those responsible for marrow engraftment following autologous bone marrow transplantation (ABMT) prompted us to establish a procedure aimed at maximally increasing the concentration of the cyclophosphamide derivative mafosfamide used for marrow purging. It, therefore, was the aim of the present study to investigate in a group of patients with acute nonlymphoblastic leukemia (ANLL; n = 19) and acute lymphoblastic leukemia (ALL; n = 19) in complete remission the effect of mafosfamide at the level of adherent blast colony-forming units (blast colony-forming units, CFU-Blast), as well as multipotential (granulocyte erythrocyte macrophage megakaryocyte colony-forming units, CFU-GEMM), erythroid (erythroid burst-forming units, BFU-E), and granulocyte-macrophage (granulocyte-macrophage colony-forming units, CFU-GM) progenitor cells. When nonadherent marrow mononuclear cells (MNCs) were incubated (30 min, 37 degrees C) with increasing doses of mafosfamide (30-120 micrograms/ml), a statistically significant (p less than or equal to 0.0005) dose-dependent suppression of CFU-Blast growth was observed. The mean (+/- 1 standard error of the mean [SEM]) values of 50% inhibition (ID50) of the CFU-Blast growth were not significantly different for ANLL (106 +/- 5) and ALL (107 +/- 5) patients. Analysis of CFU-Blast ID50 distribution demonstrated that ID50 ranged from 100 to 120 micrograms/ml in 17 cases (45%), whereas it ranged from 60 to 100 micrograms/ml in 12 cases and from 120 to 160 micrograms/ml in 9 cases. A statistically significant (p less than or equal to 0.05), dose-dependent suppression of colony growth from multi-potential and lineage-restricted progenitor cells was also observed. However, the value of CFU-Blast ID50 was significantly higher (p less than or equal to 0.05) than CFU-GEMM, BFU-E, and CFU-GM ID50 and ID95 values. In conclusion, our data demonstrate that: 1) the CFU-Blast assay allows to detect on an individual basis the doses of mafosfamide used for marrow purging, and 2) the concentrations of mafosfamide extrapolated by using the CFU-Blast assay are significantly higher than those obtained with the CFU-GM assay. The absence of any detrimental effect on marrow engraftment in vivo supports the safety of the CFU-Blast assay to evaluate the dose of mafosfamide used for marrow purging before ABMT.

Antineoplastic Agents↗

Negative regulation of granulocytic differentiation in the myeloid precursor cell line 32Dcl3 by ear-2, a mammalian homolog of Drosophila seven-up, and a chimeric leukemogenic gene, AML1/ETO.

The polyomavirus enhancer binding protein 2alphaB (AML1/PEBP2alphaB/Cbfa2) plays a pivotal role in granulocyte colony-stimulating factor (G-CSF)-mediated differentiation of a myeloid progenitor cell line, 32Dc13. In this article, we report the identification of a PEBP2alphaB interacting protein, Ear-2, an orphan member of the nuclear hormone receptor superfamily that directly binds to and can inhibit the function of PEBP2alphaB. Ear-2 is expressed in proliferating 32Dc13 cells in presence of interleukin 3 but is down-regulated during differentiation induced by G-CSF. Interestingly, AML1/ETO(MTG8), a leukemogenic chimeric protein can block the differentiation of 32Dc13 cells, which is accompanied by the sustained expression of ear-2. Overexpression of Ear-2 can prevent G-CSF-induced differentiation, strongly suggesting that ear-2 is a key negative regulator of granulocytic differentiation. Our results indicate that a dynamic balance existing between PEBP2alphaB and Ear-2 appears to determine the choice between growth or differentiation for myeloid cells.

Animals↗